Cytochalasin and isoindolinone derivatives as inhibitors of angiogenesis

ABSTRACT

The present invention relates to inhibition of angiogenesis and the treatment of diseases mediated by angiogenesis. Particularly, the invention relates to the inhibition of neovascularization and the treatment of cancer. The invention further relates to the use of cytochalasin derivatives for the inhibition of angiogenesis and the treatment of angiogenesis associated diseases. The invention also relates to new isoindolinone compounds, compositions containing them, and methods of inhibiting angiogenesis and treating angiogenesis associated diseases with the isoindolinone derivatives.

RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationSer. No. 60/041,399.

FIELD OF THE INVENTION

The invention relates generally to the inhibition of angiogenesis. Moreparticularly, the invention relates to the treatment of angiogenesisdependent and angiogenesis associated diseases, such asneovascularization and cancer. Further, the invention relates to the useof cytochalasin derivatives and isoindolinone derivatives.

BACKGROUND OF THE INVENTION

Angiogenesis is the generation of new blood vessels into a tissue ororgan. Under normal physiological conditions, humans and animals undergoangiogenesis only in very specific restricted situations. For example,angiogenesis is normally observed in wound healing, fetal and embryonaldevelopment, and formation of the corpus luteum, endometrium andplacenta.

Angiogenesis is controlled through a highly regulated system ofangiogenic stimulators and inhibitors. The control of angiogenesis hasbeen found to be altered in certain disease states and, in many cases,pathological damage associated with the diseases related to uncontrolledangiogenesis. Both controlled and uncontrolled angiogenesis are thoughtto proceed in a similar manner. Endothelial cells and pericytes,surrounded by a basement membrane, form capillary blood vessels.Angiogenesis begins with the erosion of the basement membrane by enzymesreleased by endothelial cells and leukocytes. Endothelial cells, liningthe lumen of blood vessels then protrude through the basement membrane.Angiogenic stimulants induce the endothelial cells to migrate throughthe eroded basement membrane. The migrating cells form a "sprout" offthe parent blood vessel where the endothelial cells undergo mitosis andproliferate. The endothelial sprouts merge with each other to formcapillary loops, creating a new blood vessel.

Persistent, unregulated angiogenesis occurs in a multiplicity of diseasestates, tumor metastases, and abnormal growth by endothelial cells. Thediverse pathological disease states in which unregulated angiogenesis ispresent have been grouped together as angiogenic-dependent orangiogenic-associated diseases.

One example of a disease mediated by angiogenesis is ocular neovasculardisease. This disease is characterized by invasion of new blood vesselsinto the structures of the eye, such as the retina or cornea. It is themost common cause of blindness and is involved in approximately twentyeye diseases. In age-related macular degeneration, the associated visualproblems are caused by an ingrowth of choroidal capillaries throughdefect in Bruch's membrane with proliferation of fibrovascular tissuebeneath the retinal pigment epithelium. Angiogenic damage is alsoassociated with diabetic retinopathy, retinopathy of prematurity,corneal graph rejection, neovascular glaucoma, and retrolentalfibroplasia. Other diseases associated with corneal neovascularizationinclude, but are not limited to, epidemic keratoconjunctivitis, VitaminA deficiency, contact lens overwear, atopic keratitis, superior limbickeratitis, pterygium keratitis sicca, sjogrens disease, acne rosacea,phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration,chemical burns, bacterial ulcers, fungal ulcers, Herpes simplexinfection, Herpes zoster infections, protozoan infections, Kaposi'ssarcoma, Mooren's ulcer, Terrien's marginal degeneration, marginalkeratolysis, rheumatoid arthritis, systemic lupus, polyarteritis,trauma, Wegener's sarcoidosis, scleritis, Stevens-Johnson's disease,pemphigoid, and radial keratotomy.

Diseases associated with retinal/choroidal neovascularization include,but are not limited to, diabetic retinopathy, macular degeneration,sickle cell anemia, sarcoidosis, syphilis, pseudoxanthoma elasticum,Paget's disease, vein occlusion, artery occlusion, carotid obstructivedisease, chronic uveitis/vitritis, Mycobacteria infections, lyme'sdisease, systemic lupus erythematosis, retinopathy of prematurity,Eale's disease, Behcet's disease, infections causing a retinitis orchoroiditis, presumed ocular histoplasmosis, Best's disease, myopia,optic pits, Stargardt's disease, pars planitis, chronic retinaldetachment, hyperviscosity syndromes, toxoplasmosis, trauma andpost-laser complications. Other eye-related diseases include, but arenot limited to, diseases associated with rubeosis (neovascularization ofthe angle) and diseases caused by the abnormal proliferation offibrovascular or fibrous tissue, including all forms of prolificvitreoretinopathy.

Another angiogenesis associated disease is rheumatoid arthritis. Theblood vessels in the synovial lining of the joints undergo angiogenesis.In addition to forming new vascular networks, the endothelial cellsrelease factors and reactive oxygen species that lead to pannus growthand cartilage destruction. Angiogenesis may also play a role inosteoarthritis. The activation of the chondrocytes by angiogenic-relatedfactors contributes to the destruction of the joint. At a later stage,the angiogenic factors promote new bone growth. Therapeutic interventionthat prevents the bone destruction could halt the progress of thedisease and provide relief for persons suffering with arthritis.

Chronic inflammation may also involve pathological angiogenesis. Suchdiseases as ulcerative colitis and Crohn's disease show histologicalchanges with the ingrowth of new blood vessels and the inflamed tissues.Bartonelosis, a bacterial infection found in South America, can resultin a chronic stage that is characterized by proliferation of vascularendothelial cells. Another pathological role associated withangiogenesis is found in atherosclerosis. The plaques formed within thelumen of blood vessels have been shown to have angiogenic stimulatoryactivity.

The hypothesis that tumor growth is angiogenesis-dependent was firstproposed in 1971. (Folkman, New Eng. J Med., 285:1182-86 (1971)) In itssimplest terms, this hyposthesis states: "Once tumor `take` hasoccurred, every increase in tumor cell population must be preceded by anincrease in new capillaries converging on the tumor." Tumor `take` iscurrently understood to indicate a prevascular phase of tumor growth inwhich a population of tumor cells occupying a few cubic millimetersvolume, and not exceeding a few million cells, can survive on existinghost microvessels. Expansion of tumor volume beyond this phase requiresthe induction of new capillary blood vessels. For example, pulmonarymicrometastases in the early prevascular phase in mice would beundetectable except by high power microscopy on histological sections.

Examples of the indirect evidence which support this concept include:

(1) The growth rate of tumors implanted in subcutaneous transparentchambers in mice is slow and linear before neovascularization, and rapidand nearly exponential after neovascularization. (Algire, et al., J.Nat. Cancer Inst., 6:73-85 (1945)).

(2) Tumors grown in isolated perfused organs where blood vessels do notproliferate are limited to 1-2 mm³ but expand rapidly to >1000 timesthis volume when they are transplanted to mice and becomeneovascularized. (Folkman, et al., Annals of Surgery, 164:491-502(1966)).

(3) Tumor growth in the avascular cornea proceeds slowly and at a linearrate, but switches to exponential growth after neovascularization.(Gimbrone, Jr., et al., J. Nat. Cancer Inst., 52:421-27 (1974)).

(4) Tumors suspended in the aqueous fluid of the anterior chamber of therabbit eye remain viable, avascular, and limited in size to <1 mm³. Oncethey are implanted on the iris vascular bed, they become neovascularizedand grow rapidly, reaching 16,000 times their original volume within 2weeks. (Gimbrone, Jr., et al., J. Exp. Med., 136:261-76).

(5) When tumors are implanted on the chick embryo chorioallantoicmembrane, they grow slowly during an avascular phase of >72 hours, butdo not exceed a mean diameter of 0.93+0.29 mm. Rapid tumor expansionoccurs within 24 hours after the onset of neovascularization, and by day7 these vascularized tumors reach a mean diameter of 8.0+2.5 mm.(Knighton, British J. Cancer, 35:347-56 (1977)).

(6) Vascular casts of metastases in the rabbit liver revealheterogeneity in size of the metastases, but show a relatively uniformcut-off point for the size at which vascularization is present. Tumorsare generally avascular up to 1 mm in diameter, but are neovascularizedbeyond that diameter. (Lien, et al., Surgery, 68:334-40 (1970)).

(7) In transgenic mice which develop carcinomas in the beta cells of thepancreatic islets, pre-vascular hyperplastic islets are limited in sizeto <1 mm. At 6-7 weeks of age, 4-10% of the islets becomeneovascularized, and from these islets arise large vascularized tumorsof more than 1000 times the volume of the pre-vascular islets. (Folkman,et al., Nature, 339:58-61 (1989)).

(8) A specific antibody against VEGF (vascular endothelial growthfactor) reduces microvessel density and causes "significant or dramatic"inhibition of growth of three human tumors which rely on VEGF as theirsole mediator of angiogenesis (in nude mice). The antibody does notinhibit growth of the tumor cells in vitro. (Kim, et al., Nature362:841-44 (1993)).

(9) Anti-bFGF monoclonal antibody causes 70% inhibition of growth of amouse tumor which is dependent upon secretion of bFGF as its onlymediator of angiogenesis. The antibody does not inhibit growth of thetumor cells in vitro. (Hori, et al., Cancer Res., 51:6180-84 (1991)).

(10) Intraperitoneal injection of bFGF enhances growth of a primarytumor and its metastases by stimulating growth of capillary endothelialcells in the tumor. The tumor cells themselves lack receptors for bFGF,and bFGF is not a mitogen for the tumors cells in vitro. (Gross, et al.,Proc. Am. Assoc. Cancer Res., 31:79 (1990)).

(11) A specific angiogenesis inhibitor (AGM-1470) inhibits tumor growthand metastases in vivo, but is much less active in inhibiting tumor cellproliferation in vitro. It inhibits vascular endothelial cellproliferation half-maximally at 4 logs lower concentration than itinhibits tumor cell proliferation. (Ingber, et al., Nature, 48:555-57(1990)). There is also indirect clinical evidence that tumor growth isangiogenesis dependent.

(12) Human retinoblastomas that are metastatic to the vitreous developinto avascular spheroids which are restricted to less than 1 mm³ despitethe fact that they are viable and incorporate ³ H-thymidine (whenremoved from an enucleated eye and analyzed in vitro).

(13) Carcinoma of the ovary metastasizes to the peritoneal membrane astiny avascular white seeds (1-3 mm³). These implants rarely grow largeruntil one or more of them becomes neovascularized.

(14) Intensity of neovascularization in breast cancer (Weidner, et al.,New Eng. J. Med., 324:1-8 (1991); Weidner, et al., J Nat. Cancer Inst.,84:1875-87 (1992)) and in prostate cancer (Weidner, et al., Am. JPathol., 143(2):401-09 (1993)) correlates highly with risk of futuremetastasis.

(15) Metastasis from human cutaneous melanoma is rare prior toneovascularization. The onset of neovascularization leads to increasedthickness of the lesion and an increased risk of metastasis.(Srivastava, et al., Am. J. Pathol., 133:419-23 (1988)).

(16) In bladder cancer, the urinary level of an angiogenic protein,bFGF, is a more sensitive indicator of status and extent of disease thanis cytology. (Nguyen, et al., J. Nat. Cancer Inst., 85:241-42 (1993)).

Thus, it is clear that angiogenesis plays a major role in the metastasisof cancer. If this angiogenic activity could be repressed or eliminated,then the tumor, although present, would not grow. In the disease state,prevention of angiogenesis could avert the damage caused by the invasionof the new microvascular system. Therapies directed at control of theangiogenic processes could lead to the abrogation or mitigation of thesediseases.

Angiogenesis has been associated with a number of different types ofcancer, including solid tumors and blood-borne tumors. Solid tumors withwhich angiogenesis has been associated include, but are not limited to,rhabdomyosarcomas, retinoblastoma, Ewing's sarcoma, neuroblastoma, andosteosarcoma. Angiogenesis is also associated with blood-borne tumors,such as leukemias, any of various acute or chronic neoplastic diseasesof the bone marrow in which unrestrained proliferation of white bloodcells occurs, usually accompanied by anemia, impaired blood clotting,and enlargement of the lymph nodes, liver and spleen. It is believed tothat angiogenesis plays a role in the abnormalities in the bone marrowthat give rise to leukmia-like tumors.

One of the most frequent angiogenic diseases of childhood is thehemangioma. Hemangioma is a tumor composed of newly-formed bloodvessels. In most cases the tumors are benign and regress withoutintervention. In more severe cases, the tumors progress to largecavernous and infiltrative forms and create clinical complications.Systemic forms of hemangiomas, hemangiomatoses, have a high mortalityrate. Therapy-resistant hemangiomas exist that cannot be treated withtherapeutics currently in use.

Angiogenesis is also responsible for damage found in heredity diseasessuch as Osler-Weber-Rendu disease, or heredity hemorrhagictelangiectasia. This is an inherited disease characterized by multiplesmall angiomas, tumors of blood or lymph vessels. The angiomas are foundin the skin and mucous membranes, often accompanied by epitaxis (nosebleeds) or gastrointestinal bleeding and sometimes with pulmonary orhepatitic arteriovenous fistula.

What is needed, therefore, is a composition and method which can inhibitangiogenesis. What is also needed is a composition and method which caninhibit the unwanted growth of blood vessels, especially in tumors.

Angiogenesis is also involved in normal physiological processes, such asreproduction and wound healing. Angiogenesis is an important step inovulation and also in implantation of the blastula after fertilization.Prevention of angiogenesis could be used to induce amenorrhea, to blockovulation, or to prevent implantation by the blastula.

In wound healing, excessive repair or fibroplasia can be a detrimentalside effect of surgical procedures and may be caused or exacerbated byangiogenesis. Adhesions are a frequent complication of surgery and leadto problems such as small bowel obstruction.

Several compounds have been used to inhibit angiogenesis. Taylor, et al(Nature, 297:307 (1982)) have used protamine to inhibit angiogenesis.The toxicity of protamine limits its practical use as a therapeutic.Folkman, et al. (Science, 221:719 (1983), and U.S. Pat. Nos. 5,001,116and 4,994,443) have disclosed the use of heparin and steroids to controlangiogenesis. Steroids, such as tetrahydrocortisol, which lackgluccocorticoid and mineralocorticoid activity, have been found to beangiogenic inhibitors.

Other factors found endogenously in animals, such as a 4 kDaglycoprotein from bovine vitreous humor and a cartilage derived factor,have been used to inhibit angiogenesis. Cellular factors, such asinterferon, inhibit angiogenesis. For example, interferon alpha or humaninterferon beta have been shown to inhibit tumor-induced angiogenesis inmouse dermis stimulated by human neoplastic cells. Interferon beta isalso a potent inhibitor of angiogenesis induced by allogeneic spleencells. (Sidky, et al., Cancer Res., 47:5155-61(1987)). Human recombinantinterferon (alpha/A) was reported to be successfully used in thetreatment of pulmonary hemangiomatosis, an angiogenesis-induced disease.(White, et al., New Eng. J. Med., 320:1197-1200 (1989)).

Other agents which have been used to inhibit angiogenesis includeascorbic acid ethers and related compounds. (Japanese Kokai Tokkyo KohoNo. 58-13 (1978)). Sulfated polysaccharide DS 4152 also inhibitsangiogenesis. (Japanese Kokai Tokkyo Koho No. 63-119500).

The above compounds lack adequate potency or are too toxic for practicaluse. Thus, methods and compositions are needed that are easilyadministered and capable of inhibiting angiogenesis.

Cytochalasins are secondary metabolites of mold and fungi. They areclassified into two groups, Ascomycotina and Deuteromycotina. Four othertypes of compounds are associated with cytochalasins due to theirsimilarity in chemical structure and activity. These are Chaetoglobosins(Chaetomium sp.), Aspochalasins (Aspergillus sp.), Zygosporins(Zygosporum sp.), and Phomins (Phoma sp.). The cytochalasins includefifteen compounds named from A to M (e.g., cytochalasin A); thechaetoglobulins include 13 compounds named from A to K; theaspochalasins include four compounds named from A to D. There are fivezygosporins and five phomins which are each associated with a differentcytochalasin. There are also many known derivatives of each of thevarious cytochalasins, for example, 17-hydroxycytochalasins,19,20-dihydrocytochalasins, and substituted cytochalasins.(Cytochalasins: Biochemical and Aspects, S. W. Tannenbaum, ed., NorthHolland Pub. Co., 15, 18, 320 (1978)). Reduction products ofcytochalasins are also known. (Steyn, et al., J. Chem. Soc., PerkinTrans 1, 541-44 (1982)).

Structurally, cytochalasins contain a highly substituted hydrogenatedisoindolinone ring system, called cytochalasan, to which a macrocyclicring is fused. The macrocyclic ring varies from 11 to 14 atoms in sizeand is either a carbocyclic ring, a lactone, or a cyclic carbonate. Themajor structural differences amongst cytochalasins, chaetoglobosins, andaspochalasins are a phenyl ring, indolyl group, and isopropyl grouprespectively at the C-10 position.

Cytochalasins can be produced by fermentation. For example, Adridge, etal. (J. Chem. Soc., C: 1667-76 (1967)) have suggested that cytochalasinsA and B can be produced from fermentation on Raulin's medium. Othercytochalasins can be produced from KC medium with shredded wheat.(Springer, et al., Tet. Lett., 1355-58 (1976); Zabel, et al., Appl.Environ. Microbiol., 37:208-17 (1979); Cutler, et al., J. Agric. FoodChem., 28: 139-42 (1980); Probst and Tamm, Helv. Chem. Acta., 64(7):2056-64 (1981)) Some cytochalasins have also been produced by shakeculture using glucose, soybean cake, KH₂ PO₄ and corn steep liquormedium. (Sekita, et al., Tet. Lett., 1351-54 (1976)).

Several biosynthetic pathways have been used to generate cytochalasins.For example, cytochalasin D has been synthesized from Zygosporiummasonii using ¹³ C and ¹⁴ C labelled sodium acetate, propionate, andmalonate. This process uses the acetate malonate pathway to generate aC-16 polyketide moiety in which eight acetate units are linked head totail. The polyketide is then incorporated with L-phenylalanine, followedby condensation to form a 5-membered lactum. The lactum undergoesreduction and dehydration, followed by Diels-Alder cyclization to give acytochalasin. The carbonate and lactone ring system can be formed, forexample, by Bayer-Villiger type oxidation of the macrocycle. (Binder, etal., J. Chem. Soc. Perkin Trans., 1: 1146-47 (1973); Lebet and Tamm,Helv. Chem. Acta., 57: 1785-1801 (1974); Vederas and Tamm, Helv. Chim.Acta., 59: 558-66 (1976); Vederas, et al., Helv. Chim. Acta, 58, 1886-98(1975); and Wyss, et al., Helv. Chim. Acta, 63: 1538-41 (1980)).

Another method for producing cytochalasins is Kolbe coupling. Forexample, cytochalasin B has been synthesized from (+) citronellol and(+) malic acid derivatives. (Stork, et al., J. Am. Chem. Soc., 100:7775-77 (1978)). Vedejs and Reid have also described the synthesis ofzygosporin G. (Vedejs and Reid, J. Am. Chem. Soc., 106: 4617-18 (1984)).Additionally, a number of researchers have described the partialsynthesis of cytochalasins, such as formation of the isoindolinone unitand cycloundecaconone. (Kim and Weinreb, Tet. Lett., 20: 576-82 (1979);Owen and Raphael, J. Chem. Soc. Perkin Trans., 1: 1504-07 (1978).

Cytochalasins are capable of eliciting and moderating several cellularactivities, such as enucleation of cells, inhibition of cell motility,and interference with cytoplasmic cleavage. Cytochalasins also affectthe transportation of certain biochemicals across the cell memberane.

In a study by Carter (Nature, 293: 302-5 (1967)), cytochalasinsdisplaced the nuclei from the cytoplasm of cultured L929 cells withoutaffecting cell variability. This response is dose and time dependent. Atlower doses and incubation times, the effect can be reversed bytransferring the cells to medium which does not contain cytochalasin. Athigher doses and incubatoin times, the L929 cells undergo enucleation.

The mechanism for enucleation is not known; however, several hypotheseshave been proposed. Poste and Lyon (Cytochalasins: Biochemical and CellBiological Aspects, S. W. Tannenbaum, ed., 161-89 (1978)) suggest thatenucleation occurs due to depolymerization of cortical microfilaments,along with an increase in hydrostatic pressure in the cytoplasm. Bhisey,et al. (Exp. Cell Res., 95: 376-84 (1975)) suggest that enucleationoccurs due to active contraction of cytoplasm. Their studies showed anumber of morphological changes in cell structure when the cells weretreated with cytochalasin B which indicates that enucleation is anactive, rather than passive, phenomenon.

Cytochalasins exhibit a number of cytotoxic and teratogenetic effects.These include mutinucleation, inhibition of fertilization, teratogeniceffects, and chromosomal abnormalities. Cytochalasins have been shown todepolymerize microfilaments in the contractile ring during telophase,resulting in nuclear division not followed by cytokinesis. Thus,multimucleated cells are produced. (Aubin, et al., Exp. Cell Res., 136:63-79 (1981)). The affects of cytochalasin on microfilaments alsoresults in inhibition of DNA synthesis (O'Neil, J. Cell Physio., 101:201-17 (1980)) and inhbition of cytokinesis. (Cytochalasins: Biochemicaland Cell Biological Aspects, S. W. Tannenbaum, ed., North Holland Pub.Co., 217-55 (1978); O'Neil and Renzetti, Cancer Res., 43: 521-28 (1983);Maness and Walsh, Cell, 30: 253-62 (1982)). Further, inhibition ofmicrofilaments inhibits fertilization of eggs (Brunhouse, et al., Biol.Bull., 143: 456 (1982)), formation of fertilization cone, and elongationof micro villi. (Longo, Dev. Biol., 67: 249-65 (1978); Longo, Dev.Biol., 74: 422-33 (1980); Schatten and Schatten, Dev. Biol., 78: 435-49(1980); Byrd, J. Cell Biol., 75: 267 (1977); Eddy and Shapiro, J. CellBiol., 71: 35-48 (1976)).

Cytochalasins also inhibit cell adhesion due to changes in the cellsurface. In part these changes are due to changes in the microfilamentsthat affect electrical properties in the cell membrane. (Vaidyasagar,Advances in cytochalasins, Indian Drugs Res. Assoc., 149-57 (1986)).Fluctuations in these electrical properties cause morphological changesin the cell membrane. (Wadekar, et al., Exp. Cell Biol., 48: 155-66(1980); Ghaskadbi and Mulherkar, Exp. Cell Biol., 50: 155-61 (1982)).These changes are also due in part to the action of the cytochalasins oncell surface macromolecules. This action inhibits cell growth andmotility. These changes are also due to inhibition in the synthesis ofmucopolysaccharide glycoprotein complex which binds cells together.(Sangar and Holtzer, Am. J. Anat., 135: 293-98 (1972); Burnside andManasek, Dev. Biol., 27: 443-44 (1972); Brachet and Tencer, ActaEmbryol. Exp., 1: 83-104 (1973)). Additionally, studies withcytochalasin H show that it produces disaggregation of cells resultingin inhibition of morphogenetic movements.

The teratogenic effects of cytochalasins have been studied in severaldifferent models, such as amphibians, mice, and chick embryo explants.In chick embryos cytochalasins have been shown to interfere with neuraltube closure, cardiac looping, inhibition of primary morphogenesis ofheart neural tube closure, interkinetic nuclear migration and segmentformation, disaggregation of cells, microencephaly, exencephaly, andshortening of body axis. Although the exact mechanisms causing theseteratogenic effects are not known, one proposed mechanism is through theinhibition of microtubules that are required for early differentiationin chick embryonic sensory neurons. (Cytochalasins: Biochemical and CellBiological Aspects, S. W. Tannenbaum, ed., North Holland Pub. Co.,113-42 (1978); Austin, et al., Teratology, 25: 11-18 (1982); Karfunkel,J. Exp. Zool., 181: 289-302 (1972); Greenway, et al., Proc. Soc. Exp.Biol. Med., 155: 239-42 (1977); Peter, et al., Brain Res., 42 (1): 73-81(1987); Messier and Auclair, Dev. Biol., 36: 218-23 (1974)).

Other teratogenic effects have been demonstrated in human lymphocytes,including chromosonal abnormalities. Some of the chromosomalabnormalities associated with cytochalasins include prematurechromosomal condensation, extreme extension of chromosomes, ladder-likesecondary constriction of chromosomes associated with bi- andmulti-nucleated cells.

The cytoskeleton of the cell contains microfilaments consisting mainlyof actin. Cytochalasins affect cell motility and cell shape by alteringthese microfilaments. In normal cells, they cause a shortening andsegmentation of localized masses of actin filaments. In cell morphologystudies, a generalized cell contraction was observed. Contractileproteins form actin cables condensed into masses at the base of zeioticblebs.

Cytochalasins inhibit polymerization of actin filaments from actinnuclei by inhibiting filament elongation by blocking their growingbarbed end. (Flanagan and Lin, J. Biol. Chem., 255: 835-38 (1981); Lin,et al., Biochem., Biophys. Res. Com., 122: 244-51 (1981); Casella, etal., Nature, 293: 302-5 (1981)). Several mechanisms for blocking actinfilaments have been proposed. One proposed mechanism is thatcytochalasins bind to the growing end of actin filaments and stimulateactin ATPase leading to depolymerization of actin filaments. (Brennerand Korn, J. Biol. Chem., 254: 9982-85 (1981)). Another proposedmechanism is that cytochalasins cut actin filaments into small pieces.(Morris and Tanenbaum, Nature, 287: 637-39 (1980)). A third proposedmechanism is that depolymerization and growth of actin filaments isinhibited by capping the filaments with cytochalasins, resulting incontraction of the cytoplasmic network and, ultimately, expulsion of thenuclei from the cell.

Cytochalasins affect the transportion of certain molecules across cellmembranes. These molecules include hexose, amino acids, and variousnucleosides. This transport is neither competitive nor noncompetitive(Glinsukon, et al., Toxicol. Lett., 15: 341-48 (1983); Toskulkao, etal., Nutr. Res. Int., 27: 611-18 (1983)) and is mediated by a number offunctional carriers on the cell membrane. (Yamada, et al., J Biol.Chem., 258: 9786-92 (1982)).

SUMMARY OF THE INVENTION

In accordance with the present invention, compositions and methods areprovided that are effective for modulating angiogenesis and inhibitingunwanted angiogenesis, especially angiogenesis related toneovascularization and tumor growth. The present invention comprises themodulation and inhibition of angiogenesis with cytochalasin derivatives.The term "cytochalasin derivative" means any compound having thecytochalasan structure, including, but not limited to, Cytochalasins,Chaetoglobulins, Aspochalasins, Zygosporins, and Phomins. Preferredcytochalasin derivatives are cytochalasins and chaetoglobosins having anepoxy group, a carbonate group, or both, for example, cytochalasin E.Especially preferred compounds are cytochalasins A, B, E, F, H, Q, andR; chaetoglobosins A, C, F, and K; rosellichalasin; and derivativesthereof.

The present invention also comprises new isoindolinone derivativeshaving an epoxide group, a carbonate group, or both. The invention alsocomprises compositions containing these isoindolinone derivatives, andmethods for modulating angiogenesis and inhibiting unwanted angiogenesiswith the compounds and compositions.

The present invention provides methods and compositions for treatingdiseases and processes mediated by undesired or uncontrolledangiogenesis by administering to a human or animal a compositioncomprising a cytochalasin derivative or an isoindolinone derivative in adosage sufficient to inhibit angiogenesis. The present invention isparticularly useful for treating neovascularization and tumors.Additionally, administration of a cytochalasin derivative or anisoindolinone derivative to a human or animal with prevascularizedmetastasized tumors will prevent the growth or expansion of thosetumors.

Accordingly, it is an object of the present invention to provide acomposition comprising one or more cytochalasin derivatives.

It is another object of the present invention to provide a compositioncomprising one or more cytochalasin derivatives containing an epoxidegroup, a carbonate group, or both.

It is a yet another object of the present invention to provide acomposition comprising one or more cytochalasin or chaetoglobosin.

It is a further object of the present invention to provide newisoindolinone compounds having an epoxide group, a carbonate group, orboth.

It is an object of the present invention to provide a compositioncomprising these isoindolinone derivatives which inhibit angiogenesis.

It is another object of the present invention to provide a method forinhibiting angiogenesis.

It is yet another object of the present invention to provide acomposition for inhibiting angiogenesis by oral administration of thecomposition.

It is a further object of the present invention to provide a method oftreating diseases and processes that are mediated by angiogenesis.

It is an object of the present invention to provide a composition fortreating or repressing the growth of a cancer.

It is another object of the present invention to provide a therapy forcancer that has minimal side effects.

It is yet another object of the present invention to provide a therapyfor neovascularization.

It is a further object of the present invention to provide a method andcomposition for treating diseases and processes that are mediated byangiogenesis including, but not limited to, Kaposi's sarcoma,hemangioma, solid tumors, blood borne tumors, leukemia, metastasis,telangiectasia, psoriasis, scleroderma, pyogenic granuloma, myocardialangiogenesis, Crohn's disease, plaque neovascularization, coronarycollaterals, cerebral collaterals, arteriovenous malformations, ischemiclimb angiogenesis, corneal diseases, rubeosis, neovascular glaucoma,diabetic retinopathy, proliferative vitreoretinopathy including thoseforms not associated with diabetes, retrolental fibroplasia, arthritis,diabetic neovascularization, macular degeneration, wound healing, pepticulcer, Helicobacter related diseases, fractures, keloids,vasculogenesis, hematopoiesis, ovulation, menstruation, placentation,and cat scratch fever.

These and other objects, features, and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the reaction scheme for preparing isoindolinonederivatives of the present invention.

FIGS. 2A-2C depict the inhibition of bovine capillary endothelial cellproliferation (BCE) in Swiss mouse embryo fibroblast cells.

FIG. 3 compares the activity of cytochalasin E and epoxycytochalasin Jin the inhibition of bovine capillary endothelial cell proliferation(BCE).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In one aspect, the present invention comprises compositions and methodsof inhibiting angiogenesis with cytochalasin derivatives. The presentinvention also comprises methods of treating angiogenesis dependent andangiogenesis associated diseases by administering a cytochalasinderivative or a composition containing a cytochalasin derivative to aperson or animal having such a disease.

The present invention encompasses the use of any cytochalasinderivative. The term "cytochalasin derivative" means any compound havingthe cytochalasan structure, ##STR1## including, but not limited to,Cytochalasins, Chaetoglobulins, Aspochalasins, Zygosporins, and Phominsand derivatives thereof.

Preferred compounds of the invention include cytochalasin derivativeshaving either an epoxide group, a carbonate group, or both. Severalnaturally occurring cytochalasin derivatives contain an epoxide group.These include, but not limited to, cytochalasin E((6S,7S,16R,18R)-6,7-epoxy-18-hydroxy-16-methyl-10-phenyl-21,23-dioxa-[13]cytochalasa-13(E),19(E)-diene-1,17,22-trione),cytochalasin F((6S,7S,16R,20R)-6,7-epoxy-20-hydroxy-16-methyl-10-phenyl-24-oxa-[14]cytochalasa-13(E),21(E)-diene-1,23-dione),cytochalasin Q, cytochalasin R, rosellichalasin, and chaetoglobosin A((6S,7S,16R,19R)-6,7-epoxy-10-(indol-3-yl)-19-hydroxy-16,18-dimethyl-[13]cytochalasa-13(E),17(E),21(E)-triene-1,20,23-trione),chaetoglobosin C((6S,7S,16S)-6,7-epoxy-10-(indol-3-yl)-16,18-dimethyl-[13]cytochalasa-13(E),17(E)-diene-1,19,20,23-tetraone),chaetoglobosin F((6S,7S,16R)-6,7-epoxy-20-hydroxy-10-(indol-3-yl)-16,18-dimethyl-[13]cytochalasa-13(E),17(E)-diene-1,19,23-trione),and chaetoglobosin K. ##STR2##

The cytochalasins of the invention can be produced by fermentation frommolds or fungi by methods known in the prior art. For example,cytochalasin E is a metabolite of both Rosellinia necatrix andAspergillus clavatus (Aldridge, et al., J. Chem. Soc. (D), Chem. Comm.,148-49 (1972)), and cytochalasin D is a metabolite of Zygosporiummasonii (Vederas and Tamm, Helv. Chim. Acta, 59: 558-66 (1976)).

The present invention also comprises derivatives of these compoundswhich retain an epoxide group, a carbonate group, or both, such as17-hydroxy-cytochalasin E, 19,20-dihydro-cytochalasin E, reductionproducts of cytochalasin E, and the corresponding derivatives of othercytochalasins. Examples of these compounds include, but are not limitedto, the following: ##STR3##

These compounds can be produced by methods known in the art; forexample, those methods described in Steyn, et al. J. Chem. Soc. PerkinsTrans. 1, 541-44 (1982) and Aldridge, et al., J. Chem. Soc. Chem. Comm.,551-52 (1973).

Other cytochalasin compounds, although not naturally containing anepoxide group, can be modified to contain an epoxide group. For example,an epoxide group can be formed on cytochalasin derivatives having amethyl group attached to the benzene portion of the isoindolinone ringthrough reaction with a peroxide.

The present inventors have found that inhibition of angiogenesis isgreater in cytochalasin compounds having an epoxide group and/or acarbonate group, such as cytochalasin E. In another aspect, the presentinvention comprises new isoindolinone derivatives having an epoxidegroup, a carbonate group, or both. These compounds lack the macrocyclicring of the cytochalasin derivatives and have the following genericformula: ##STR4## wherein R is H, OH, OR₁, wherein R₁ is H, alkyl, aryl,C(O)--R₁, or C(O)OR₁ ; R₂ is H, alkyl, aryl, heteroaryl, aralkyl, orheteroaralkyl; R₃ is ═CH₂, H, CH₃ ; R₄ is H, OH, or OR₁ ;

and where there is optionally an exocyclic epoxide ring between C6 andC7, provided that the compound contains at least one epoxide orcarbonate group.

Aryl includes moieties having a 5 to 12 membered ring system, forexample phenyl and napthyl. Aralkyl include moieties having a 5 to 12membered ring system and a straight or branched alkyl chain of 1 to 8carbon atoms, for example benzyl. Heteroaryl includes moieties having a6 to 12 membered ring system containing from 1 to 3 heterocyclic atomsselected from N, O, and S. Heteroaralkyl includes moieties having a 6 to12 membered ring system containing from 1 to 3 heterocyclic atomsselected from N, O, and S and a straight or branched alkyl chain of 1 to8 carbon atoms. Each of the above identified substituents may besubstituted or unsubstituted.

Particularly preferred compounds of the present invention include thefollowing: ##STR5## These compounds have lower cytotoxicity than thecytochalasins due to the absence of the macrocyclic ring structure,allowing higher doses of the compounds to be administered withoutadverse effects. These isoindolinone derivatives also possess greaterangiogenesis inhibitory activity than the cytochalasins, allowingsmaller doses to achieve the same inhibitory activity.

The isoindolinone derivatives of the present invention can be producedfrom 6-methyl-isoindol-1,3-dione. (FIG. 1) The6-methyl-isoindol-1,3-dione first undergoes a reduction of the oxogroup. This reduction may be metal catalyzed, for example, by zinc.Next, the intermediate is optionally alkylated at the 3-position, forexample, with n-butyl lithium and benzyl bromide.

For those compounds containing an epoxide ring, the epoxide ring isformed in two steps. First, a 6-methyl-isoindol-1,3-dione derivative isreacted with a 1:1 ratio of tetrahydrofuran (THF) and tert-butyl alcoholin liquid ammonia. Next, the intermediate is reacted with a peroxy acid.The 6-methyl-isoindol-1,3-dione derivative can be carbonated byhydroboration followed by oxidation.

The present invention also comprises compositions and methods ofinhibiting angiogenesis with such isoindolinone derivatives. Theinvention further comprises methods of treating angiogenesis dependentand angiogenesis associated diseases by administering an isoindolinonederivative or a composition containing a isoindolinone derivative to aperson or animal having such a disease.

The cytochalasin and isoindolinone derivatives described above can beprovided as pharmaceutically acceptable compositions using formulationmethods known to those of ordinary skill in the art. These formulationscan be administered by standard routes. In general, the pharmaceuticalcompositions of the present invention can be administered by topical,transdermal, oral, rectal or parenteral (e.g., intravenous, subcutaneousor intramuscular) routes. In addition, the compositions can beincorporated into biodegradable polymers allowing for sustained releaseof the compound, the polymers being implanted near the desired site ofdrug delivery, for example, at the site of a tumor. Such biodegradablepolymers and their use for delivery of pharmaceuticals are known in theart. (e.g., Brem et al., J. Neurosurg. 74:441-46 (1991)).

The dosage of the compound will depend on the condition being treated,the particular compound, and other clinical factors such as the weightand condition of the human or animal to be treated and the chosen routeof administration. It is to be understood that the present invention hasapplication for both human and veterinary use. In one embodiment, thecytochalasin derivatives of the present invention can be administered tohumans at a dose between approximately 0.01 mg/kg body weight per dayand approximately 100 mg/kg body weight per day. The preferred dosage ofcytochalasin derivatives is approximately 2 mg/kg/day. The isoindolinonederivatives of the present invention can be administered at similardosages.

The pharmaceutical compositions of the present invention include thosesuitable for oral, rectal, ophthalmic, (including intravitreal orintracameral) nasal, topical (including buccal and sublingual), vaginalor parenteral (including subcutaneous, intramuscular, intravenous,intradermal, intratracheal, and epidural) administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by conventional pharmaceutical techniques. Such techniquesinclude the step of bringing into association the active ingredient andthe pharmaceutical carrier(s). In general, the formulations are preparedby uniformly and intimately bringing into associate the activeingredient with liquid carriers or finely divided solid carriers orboth, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil emulsion and as a bolus, etc.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface active ordispersing agent. Molded tablets may be made by molding, in a suitablemachine, a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may be optionally coated or scored and maybe formulated so as to provide a slow or controlled release of theactive ingredient therein.

Formulations suitable for topical administration in the mouth includelozenges comprising the ingredients in a flavored basis, usually sucroseand acacia or tragacanth; pastilles comprising the active ingredient inan inert basis such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the ingredient to be administered in a suitableliquid carrier.

Formulations suitable for topical administration to the skin may bepresented as ointments, creams, gels, and pastes comprising theingredient to be administered in a pharmaceutical acceptable carrier. Apreferred topical delivery system is a transdermal patch containing theingredient to be administered.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of 20 to 500 microns which is administered in the manner inwhich snuff is administered, i.e., by rapid inhalation through the nasalpassage from a container of the powder held close up to the nose.Suitable formulations, wherein the carrier is a liquid, foradministration, as for example, a nasal spray or as nasal drops, includeaqueous or oily solutions of the active ingredient.

Formulations suitable for vaginal administration may be presented aspessaries, tamports, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats, and solutes which render the formulationisotonic with the blood of the intended recipient and aqueous andnon-aqueous sterile suspensions which may include suspending agents andthickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example, sealed ampules and vials, and may bestored in a freeze-dried (lyophilized) conditions requiring only theaddition of the sterile liquid carrier, for example, water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules andtablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of the administered ingredient.

It should be understood that in addition to the ingredients,particularly mentioned above, the formulations of the present inventionmay include other agents conventional in the art having regard to thetype of formulation in question, for example, those suitable for oraladministration may include flavoring agents.

Diseases associated with corneal neovascularization that can be treatedaccording to the present invention include but are not limited to,diabetic retinopathy, retinopathy of prematurity, corneal graftrejection, neovascular glaucoma and retrolental fibroplasia, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalulcers, Herpes simplex infections, Herpes zoster infections, protozoaninfections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginaldegeneration, mariginal keratolysis, trauma, rheumatoid arthritis,systemic lupus, polyarteritis, Wegener's sarcoidosis, scleritis,Stevens-Johnson disease, pemphigoid radial keratotomy, and corneal graphrejection.

Diseases associated with retinal/choroidal neovascularization that canbe treated according to the present invention include, but are notlimited to, diabetic retinopathy, macular degeneration, sickle cellanemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget's disease,vein occlusion, artery occlusion, carotid obstructive disease, chronicuveitis/vitritis, mycobacterial infections, Lyme's disease, systemiclupus erythematosis, retinopathy of prematurity, Eales' disease,Behcet's disease, infections causing a retinitis or choroiditis,presumed ocular histoplasmosis, Bests disease, myopia, optic pits,Stargardt's disease, pars planitis, chronic retinal detachment,hyperviscosity syndromes, toxoplasmosis, trauma and post-lasercomplications. Other diseases include, but are not limited to, diseasesassociated with rubeosis (neovasculariation of the angle) and diseasescaused by the abnormal proliferation of fibrovascular or fibrous tissueincluding all forms of proliferative vitreoretinopathy, whether or notassociated with diabetes.

Diseases associated with chronic inflammation can be treated by thecompositions and methods of the present invention. Diseases withsymptoms of chronic inflammation include inflammatory bowel diseasessuch as Crohn's disease, ulcerative colitis, psoriasis, sarcoidosis andrheumatoid arthritis. Angiogenesis is a key element that these chronicinflammatory diseases have in common. The chronic inflammation dependson continuous formation of capillary sprouts to maintain an influx ofinflammatory cells. The influx and presence of the inflammatory cellsproduce granulomas and thus, maintains the chronic inflammatory state.Inhibition of angiogenesis by the compositions and methods of thepresent invention inhibit the formation of the granulomas and alleviatethe disease.

The compositions and methods of the present invention can be used totreat patients with inflammatory bowel diseases such as Crohn's diseaseand ulcerative colitis. Both Crohn's disease and ulcerative colitis arecharacterized by chronic inflammation and angiogenesis at various sitesin the gastrointestinal tract. Crohn's disease is characterized bychronic granulomatous inflammation throughout the gastrointestinal tractconsisting of new capillary sprouts surrounded by a cylinder ofinflammatory cells. Inhibition of angiogenesis by the compositions andmethods of the present invention inhibits the formation of the sproutsand prevents the formation of granulomas.

Crohn's disease occurs as a chronic transmural inflammatory disease thatmost commonly affects the distal ileum and colon but may also occur inany part of the gastrointestinal tract from the mouth to the anus andperianal area. Patients with Crohn's disease generally have chronicdiarrhea associated with abdominal pain, fever, anorexia, weight lossand abdominal swelling. Ulcerative colitis is also a chronic,nonspecific, inflammatory and ulcerative disease arising in the colonicmucosa and is characterized by the presence of bloody diarrhea.

The inflammatory bowel diseases also show extraintestinal manifestationssuch as skin lesions. Such lesions are characterized by inflammation andangiogenesis and can occur at many sites other than the gastrointestinaltract. The compositions and methods of the present invention are alsocapable of treating these lesions by preventing the angiogenesis, thusreducing the influx of inflammatory cells and the lesion formation.

Sarcoidosis is another chronic inflammatory disease that ischaracterized as a multisystem granulomatous disorder. The granulomas ofthis disease may form anywhere in the body and thus the symptoms dependon the site of the granulomas and whether the disease active. Thegranulomas are created by the angiogenic capillary sprouts providing aconstant supply of inflammatory cells.

The compositions and methods of the present invention can also treat thechronic inflammatory conditions associated with psoriasis. Psoriasis, askin disease, is another chronic and recurrent disease that ischaracterized by papules and plaques of various sizes. Prevention of theformation of the new blood vessels necessary to maintain thecharacteristic lesions leads to relief from the symptoms.

Another disease which can be treated according to the present inventionis rheumatoid arthritis. Rheumatoid arthritis is a chronic inflammatorydisease characterized by nonspecific inflammation of the peripheraljoints. It is believed that the blood vessels in the synovial lining ofthe joints undergo angiogenesis. In addition to forming new vascularnetworks, the endothelial cells release factors and reactive oxygenspecies that lead to pannus growth and cartilage destruction. Thefactors involved in angiogenesis may actively contribute to, and helpmaintain, the chronically inflamed state of rheumatoid arthritis. Otherdiseases that can be treated according to the present invention arehemangiomas, Osler-Weber-Rendu disease, or hereditary hemorrhagictelangiectasia, solid or blood borne tumors and acquired immunedeficiency syndrome. In particular, the invention is useful for treatingcancers, including, but not limited to, those cancers exhibiting solidtumors, such as breast, lung, ovarian, testicular, and colon cancers

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLES Example 1

Bovine capillary endothelial (BCE) cells and Swiss mouse embryofibroblast cells (NIH3T3) were isolated as previously described(Folkman, , et al., Proc. Nat. Acad. Sci USA., 76:5217-21 (1979)) andmaintained in DMEM supplemented with 10% heat-inactivated bovine calfserum (BCS), antibiotics, and 3 ng/ml recombinant human bFGF (SciosNova, Mountainview, Calif.). Monolayers of the cells growing in 6-wellplates were dispersed in a 0.05% trypsin solution. The cells werere-suspended with DMEM containing 10% BCS. Approximately 12,500 cells in0.5 ml were added to each well of gelatinized 24-well tissue cultureplates and incubated at 37° C. (in 10% CO₂) for 24 hours. The medium wasreplaced with 500 ml of fresh DMEM containing 5% BCS. Samples ofcytochalasin E, cytochalasin A, and cytochalasin H were added to eachwell in triplicate. After 30 minutes of incubation, bFGF was added to afinal concentration of 1 ng/ml. After 72 hours of incubation, cells weretrypsinized, re-suspended in Hematall (Fisher Scientific, Pittsburg,Pa.) and counted with a Coulter counter.

The results are shown in FIG. 2. FIG. 2 is a graph showing theinhibition of bovine capillary endothelial cell proliferation (BCE)(squares) compared to Swiss mouse embryo fibroblast cells (NIH3T3). Theabscissa shows absorbance at 630 Angstroms by stained cell nuclei, whichcorresponds to cell number. The ordinate measures drug concentration ingrams per milliliter. FIG. 2A shows the administration of cytochalasinE; FIG. 2B shows the administration of cytochalasin H, and the FIG. 2Cshows the administration of cytochalasin A.

The results show that cytochalasin E inhibits endothelial cellproliferation with an IC₅₀ of 2 pm/ml, and is 40,000/l selective forendothelial cells over fibroblast cells. The other two cytochalasins,cytochalasin H and cytochalasin A, which lack epoxide moieties, are1000-fold less active.

Example 2

Bovine capillary endothelial (BCE) were isolated as previously described(Folkman, , et al., Proc. Nat. Acad. Sci USA., 76:5217-21 (1979)) andmaintained in DMEM supplemented with 10% heat-inactivated bovine calfserum (BCS), antibiotics, and 3 ng/ml recombinant human bFGF (SciosNova, Mountainview, Calif.). Monolayers of the cells growing in 6-wellplates were dispersed in a 0.05% trypsin solution. The cells werere-suspended with DMEM containing 10% BCS. Approximately 12,500 cells in0.5 ml were added to each well of gelatinized 24-well tissue cultureplates and incubated at 37° C. (in 10% CO₂) for 24 hours. The medium wasreplaced with 500 ml of fresh DMEM containing 5% BCS. Samples ofcytochalasin E and epoxycytochalasin J were added to each well intriplicate. After 30 minutes of incubation, bFGF was added to a finalconcentration of 1 ng/ml. After 72 hours of incubation, cells weretrypsinized, re-suspended in Hematall (Fisher Scientific, Pittsburg,Pa.) and counted with a Coulter counter.

The results are shown in FIG. 3. FIG. 3 shows that epoxycytochalasin Jis an even more effective inhibitor of bovine capillary endothelial cellproliferation (BCE) (squares) than cytochalasin E. The abscissa showsabsorbance at 630 Angstroms by stained cell nuclei, which corresponds tocell number. The ordinate measures drug concentration in grams permilliliter.

Example 3

Pellets for implantation into rabbit corneas were made by mixing 110 μlof saline containing 12 μg of recombinant bFGF (TakedaPharmaceuticals-Japan) with 40 mg of sucralfate (Bukh Meditec-Denmark);this suspension was added to 80 μl of 12% hydron (Interferon Sciences)in ethanol. 10 μl aliquots of this mixture was then pipetted onto teflonpegs and allowed to dry producing approximately 17 pellets. A pellet wasimplanted into corneal micropockets of each eye of an anesthetizedfemale New Zealand white rabbit, 2 mm from the limbus followed bytopical application of erythromycin ointment onto the surface of thecornea. The animals were fed daily from 2 days post-implantation bygastric lavage with cytochalasin E or the aldehyde methyl esterderivative of cytochalasin E suspended in 0.5% carboxymethyl celluloseor 0.5% carboxymethyl cellulose alone. The animals were examined with aslit lamp every other day in a masked manner by the same cornealspecialist. The area of corneal neovascularization was determined bymeasuring with a reticule the vessel length (L) from the limbus and thenumber of clock hours (C) of limbus involved. A formula was used todetermine the area of a circular band segment: C/12 * 3.1416 [r² -(r-L)²] where r=6 mm the measured radius of the rabbit cornea. Variousmathematical models were utilized to determine the amount ofvascularized cornea and this formula was found to provide the mostaccurate approximation of the area of the band of neovascularizationthat grows towards the pellet.

It is important to note that the rabbit cornea assay is preferablebecause it will generally recognize compounds that are inactive per sebut are metabolized to yield active compounds.

It was found that subcutaneously administered cytochalasin E at a dosageof 2 mg/kg/day inhibits basic fibroblast growth factor (bFGF) drivencorneal neovascularization by 50% and that the aldehyde methyl esterderivative of cytochalasin E at a dosage of 2 mg/kg/day inhibits bFGFdriven corneal neovascularization by 38%.

Example 4

The neovascularization experiment described in Example 2 was repeatedusing the corneal micropockets in mice. The compounds tested werecytochalasin E, astaphiatin, arglabin, epoxycytochalasin-H, ozonolyzedcytochalasin-E, and precursor cytochalasin-E. The results of thesecompound's ability to inhibit angiogenesis are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Com-               %        % Weight                                            pound Inhibition Loss                                                       ______________________________________                                        Cytochalasin-E     45.00    0.00                                                Astaphiatin 33.56 6.92                                                        Arglabin 22.72 2.69                                                           Epoxycytochalasin-H 27.35 2.46                                                Ozonolyzed Cytochalasin-E 20.00 4.00                                          (JHS 199)                                                                     Precursor Cytochalasin-E 20.00 2.50                                           (JHS-F6P2)                                                                  ______________________________________                                    

Example 5

By screening a variety of murine tumors capable of inhibiting their ownmetastases, a Lewis lung carcinoma was selected in which the primarytumor most efficiently inhibited lung metastasis. Syngeneic C57BI6/Jsix-week-old male mice were injected (subcutaneous dorsum) with 1×10⁶tumor cells. Visible tumors first appeared after 3-4 days. When tumorswere approximately 1500 mm³ in size, mice were randomized into twogroups. The primary tumor was completely excised in the first group andleft intact in the second group after a sham operation. Although tumorsfrom 500 mm³ to 3000 mm³ inhibited growth of metastases, 1500 mm³ wasthe largest primary tumor that could be safely resected with highsurvival and no local recurrence.

After 21 days, all mice were sacrificed and autopsied. In mice with anintact primary tumor, there were four +2 visible metastases, compared tofifty +5 metastases in the mice in which the tumor had been removed(p<0.0001). These data were confirmed by lung weight, which correlatesclosely with tumor burden, as has been previously demonstrated. Therewas a 400% increase in wet lung weight in the mice that had their tumorsremoved compared to mice in which the tumor remained intact (p<0.0001).

This experimental model gave reproducible data and the experimentdescribed is reproducible. This tumor is labeled "Lewis lungcarcinoma--low metastatic" (LLC--Low). The tumor also suppressedmetastases in a nearly identical pattern in SCID mice, which aredeficient in both B and T lymphocytes.

It was found that 2 mg/kg/day of cytochalasin E inhibited tumor growthby 74%. In the same model, it was found that 2 mg/kg/day of the aldehydemethyl ester derivative of cytochalasin E inhibited tumor growth by 30%.

Example 6

The Lewis lung experiment of Example 5 was repeated using cytochalasin Eand methanolyzed cytochalasin E. The results of this experiment arepresented in the table below.

    ______________________________________                                        Inhibition of Tumor Growth                                                        Compound            Dose      T/C                                         ______________________________________                                        Cytochalasin-E      2.0 mg/kg qd                                                                            0.28                                              Methanolyzed Cytochalasin-E 2.0 mg/kg od 0.70                               ______________________________________                                    

The above examples are intended to be demonstrative, rather thanlimiting, of the embodiments contemplated by the invention andencompassed within the scope of the claims.

We claim:
 1. A method of inhibiting angiogenesis in a human or animalcomprising administering to the human or animal an angiogenesisinhibitory amount of a composition comprising a cytochalasin derivative.2. The method of claim 1 wherein the cytochalasin derivative possessesan epoxide group, a carbonate group, or both.
 3. The method of claim 1wherein the cytochalasin derivative is selected from the groupconsisting of cytochalasin A, cytochalasin B, cytochalasin C,cytochalasin D, cytochalasin F, cytochalasin H, cytochalasin J,cytochalasin K, cytochalasin Q, cytochalasin R, rosellichalasin,epoxycytochalasin H, epoxycytochalasin J, chaetoglobosin A,chaetoglobosin C, chaetoglobosin F, and chaetoglobosin K.
 4. The methodof claim 1 wherein the cytochalasin derivative is selected from thegroup consisting of astaphiatin, arglabin, ozonolyzed cytochalasin E,methanolyzed cytochalasin E, aldehyde methyl ester of cytochalasin E,17-hydroxycytochalasin E, 19,20-dihydrocytochalasin E, and rearrangedcytochalasin E.
 5. A method of treating an angiogenesis-associated orangiogenesis-dependent disease in a human or animal comprisingadministering to the human or animal an angiogenesis inhibiting amountof a cytochalasin derivative.
 6. The method of claim 5 wherein thecytochalasin derivative possesses an epoxide group, a carbonate group,or both.
 7. The method of claim 5 wherein the cytochalasin derivative isselected from the group consisting of cytochalasin A, cytochalasin B,cytochalasin C, cytochalasin D, cytochalasin F, cytochalasin H,cytochalasin J, cytochalasin K, cytochalasin Q, cytochalasin R,rosellichalasin, epoxycytochalasin H, epoxycytochalasin J,chaetoglobosin A, chaetoglobosin C, chaetoglobosin F, and chaetoglobosinK.
 8. The method of claim 5 wherein the cytochalasin derivative isselected from the group consisting of astaphiatin, argiabin, ozonolyzedcytochalasin E, methanolyzed cytochalasin E, aldehyde methyl ester ofcytochalasin E, 17-hydroxycytochalasin E, 19,20-dihydrocytochalasin E,and rearranged cytochalasin E.
 9. A compound having the formula ##STR6##wherein R is H, OH, OR₁, wherein R₁ is H, alkyl, aryl, COH, C(O)-alkyl,C(O)-aryl, C(O)OH, C(O)O-alkyl, C(O)O-arylR₂ is H, alkyl, aryl,heteroaryl, aralkyl, or heteroaralkyl; R₃ is ═CH₂, H, CH₃ ; R₄ is H, OH,or OR₁ ;and where there is optionally an exocyclic epoxide ring betweenC6 and C7, provided that the compound contains at least one epoxide orcarbonate group.
 10. The compound of claim 9 wherein aryl is asubstituted or unsubstituted 5 to 12 membered aromatic ringsystem;aralkyl is a substituted or unsubstituted 5 to 12 memberedaromatic ring system attached through a straight or branched alkyl chainof 1 to 8 carbon atoms; heteroaryl is a substituted or unsubstituted 6to 12 membered heteroaromatic ring system containing from 1 to 3heterocyclic atoms selected from N, O, and S; and heteroaralkyl is asubstituted or unsubstituted 6 to 12 membered heteroaromatic ring systemcontaining from 1 to 3 heterocyclic atoms selected from N, O, and Sattached through a straight or branched alkyl chain 1 to 8 carbon atoms.11. The compound of claim 9 selected from the group of ##STR7##
 12. Amethod of inhibiting angiogenesis in a human or animal comprisingadministering to the human or animal an angiogenesis inhibitory amountof a composition comprising a compound having the formula whereinR is H,OH, OR₁, wherein R₁ is H, alkyl, aryl, COH, C(O)-alkyl, C(O)-aryl,C(O)OH, C(O)O-alkyl, C(O)O-aryl R₂ is H, alkyl, aryl, heteroaryl,aralkyl, or heteroaralkyl; R₃ is ═CH₂, H, CH₃ ; R₄ is H, OH, or OR₁ ;andwhere there is optionally an exocyclic epoxide ring between C6 and C7,provided that the compound contains at least one epoxide or carbonategroup.
 13. The method of claim 12 wherein the composition comprises acompound selected from the group consisting of ##STR8##
 14. A method oftreating an angiogenesis-associated or anglogenesis-dependent disease ina human or animal comprising administering to the human or animal ananglogenesis inhibiting amount of a compound of the formula whereinR isH, OH, OR₁, wherein R₁ is H, alkyl, aryl, COH, C(O)-alkyl, C(O)-aryl,C(O)OH, C(O)O-alkyl, C(O)O-aryl R₂ is H, alkyl, aryl, heteroaryl,aralkyl, or heteroaralkyl; R₃ is ═CH₂, H, CH₃ ; R₄ is OH, or OR₁ ;andwhere there is optionally an exocyclic epoxide ring between C6 and C7,provided that the compound contains at least one epoxide or carbonategroup.
 15. The method of claim 14 wherein the composition comprises acompound selected from the group consisting of ##STR9##